This project studies human immunodeficiency virus type 1 (HIV-1) genetic variability, particularly in the env gene, and how it affects recognition by the immune system of the host. This is of particular importance in considering vaccines. One focus of study is to identify env determinants recognized by neutralizing antibodies and how variability alters neutralizing activity. Transmission of molecularly cloned viruses in the presence of neutralizing antibodies results in the appearance of mutants that resist neutralization by the selecting antisera. Several mutants have been identified to date which identify distinct natural human neutralizing antibodies of broad specificity, and evidence suggests that these recognize conformational rather than sequential epitopes. Studies have been directed toward the relative importance of the disulfide loop containing the third hypervariable region (V3), which is an immunodominant determinant in infected people and is a neutralization target in serum from hyperimmunized animals, in neutralization by human sera. Using an infectious clone of hybrid HIV-1(IIIB) containing heterologous V3 loops, neutralization by hyperimmune sera depends primarily on the sequence of the V3 loop and is highly type-specific, but with human serum depends in part on envelope conformation and is more broadly reactive. Studies of HIV-1 intrapatient variation over time and of interpatient variation within populations have also given insights into the relationship of variation and virus neutralization. To better study neutralizing antibodies, immunoglobulin libraries from B cells of HIV-1-infected people have been obtained in bacterial expression vectors. These are currently being screened for recognition of viral proteins and neutralizing activity. Neutralizing antibodies from such libraries should allow a much better understanding of the basis for neutralization of HIV-1.